Indicators of HSV1 Infection, ECM-Receptor Interaction, and Chromatin Modulation in a Nuclear Family with Schizophrenia.

Schizophrenia (SCZ) is a complex psychiatric disorder with high heritability; identifying risk genes is essential for deciphering the disorder's pathogenesis and developing novel treatments. Using whole-exome sequencing, we screened for mutations within protein-coding sequences in a single family of patients with SCZ. In a pathway enrichment analysis, we found multiple transmitted variant genes associated with two KEGG pathways: herpes simplex virus 1 (HSV1) infection and the extracellular matrix (ECM)-receptor interaction. When searching for rare variants, six variants, SLC6A19p.L541R, CYP2E1p.T376S, NAT10p.E811D, N4BP1p.L7V, CBX2p.S520C, and ZNF460p.K190E, segregated with SCZ. A bioinformatic analysis showed that three of these mutated genes were associated with chromatin modulation. We found that HSV1 infection, ECM-receptor interaction pathways, and epigenetic mechanisms may contribute to the pathogenesis of SCZ in certain families. The identified polygenetic risk factors from the sample family provide distinctive underlying biological mechanisms of the pathophysiology of SCZ and may be useful in clinical practice and patient care.

Identification of rare missense mutations in the glutamate ionotropic receptor AMPA type subunit genes in schizophrenia.

OBJECTIVE: The alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors significantly regulate the synaptic transmission and functions of various synaptic receptors. This study aimed to identify single nucleotide mutations in the glutamate receptor, ionotropic, AMPA type (GRIA) gene family, which is associated with schizophrenia. METHODS: The exon regions of four genes (GRIA1, GRIA2, GRIA3, and GRIA4) encoding glutamate ionotropic receptor AMPA type proteins were resequenced in 516 patients with schizophrenia. We analyzed the protein function of the identified rare mutants via immunoblotting. RESULTS: A total of 24 coding variants were detected in the GRIA gene family, including six missense mutations, 17 synonymous mutations, and one frameshift insertion. Notably, three ultra-rare missense mutations (GRIA1p.V182A, GRIA2p.P123Q, and GRIA4p.Y491H) were not documented in the single nucleotide polymorphism database, gnomAD genomes, and 1517 healthy controls available from Taiwan BioBank. Immunoblotting revealed GRIA4p.Y491H mutant with altered protein expressions in cultured cells compared with the wild type. CONCLUSION: Our findings suggest that, in some patients affected by schizophrenia, the GRIA gene family harbors rare functional mutations, which support rare coding variants that could contribute to the genetic architecture of this illness. The in-vitro impacts of these rare pathological mutations on the pathophysiology of schizophrenia are worthy of future investigation.

Ultrarare Loss-of-Function Mutations in the Genes Encoding the Ionotropic Glutamate Receptors of Kainate Subtypes Associated with Schizophrenia Disrupt the Interaction with PSD95.

Schizophrenia is a complex mental disorder with a genetic component. The GRIK gene family encodes ionotropic glutamate receptors of the kainate subtype, which are considered candidate genes for schizophrenia. We screened for rare and pathogenic mutations in the protein-coding sequences of the GRIK gene family in 516 unrelated patients with schizophrenia using the ion semiconductor sequencing method. We identified 44 protein-altered variants, and in silico analysis indicated that 36 of these mutations were rare and damaging or pathological based on putative protein function. Notably, we identified four truncating mutations, including two frameshift deletion mutations (GRIK1p.Phe24fs and GRIK1p.Thr882fs) and two nonsense mutations (GRIK2p.Arg300Ter and GRIK4p.Gln342Ter) in four unrelated patients with schizophrenia. They exhibited minor allele frequencies of less than 0.01% and were absent in 1517 healthy controls from Taiwan Biobank. Functional analysis identified these four truncating mutants as loss-of-function (LoF) mutants in HEK-293 cells. We also showed that three mutations (GRIK1p.Phe24fs, GRIK1p.Thr882fs, and GRIK2p.Arg300Ter) weakened the interaction with the PSD95 protein. The results suggest that the GRIK gene family harbors ultrarare LoF mutations in some patients with schizophrenia. The identification of proteins that interact with the kainate receptors will be essential to determine kainate receptor-mediated signaling in the brain.

Transcriptomic and Proteomic Analysis of CRISPR/Cas9-Mediated ARC-Knockout HEK293 Cells.

Arc/Arg3.1 (activity-regulated cytoskeletal-associated protein (ARC)) is a critical regulator of long-term synaptic plasticity and is involved in the pathophysiology of schizophrenia. The functions and mechanisms of human ARC action are poorly understood and worthy of further investigation. To investigate the function of the ARC gene in vitro, we generated an ARC-knockout (KO) HEK293 cell line via CRISPR/Cas9-mediated gene editing and conducted RNA sequencing and label-free LC-MS/MS analysis to identify the differentially expressed genes and proteins in isogenic ARC-KO HEK293 cells. Furthermore, we used bioluminescence resonance energy transfer (BRET) assays to detect interactions between the ARC protein and differentially expressed proteins. Genetic deletion of ARC disturbed multiple genes involved in the extracellular matrix and synaptic membrane. Seven proteins (HSPA1A, ENO1, VCP, HMGCS1, ALDH1B1, FSCN1, and HINT2) were found to be differentially expressed between ARC-KO cells and ARC wild-type cells. BRET assay results showed that ARC interacted with PSD95 and HSPA1A. Overall, we found that ARC regulates the differential expression of genes involved in the extracellular matrix, synaptic membrane, and heat shock protein family. The transcriptomic and proteomic profiles of ARC-KO HEK293 cells presented here provide new evidence for the mechanisms underlying the effects of ARC and molecular pathways involved in schizophrenia pathophysiology.

Sequencing of the coding regions of GNBIL on chromosome 22q11.2 as a risk gene of schizophrenia.

GNB1L haploinsufficiency caused by 22q11.2 deletion syndrome may contribute to schizophrenia pathophysiology. We resequenced the protein-coding sequences of GNB1L in 553 patients with schizophrenia and 535 controls from Taiwan. Four common single-nucleotide polymorphisms showed no association with patients with schizophrenia. We identified 17 rare missense mutations, including three that were schizophrenia-associated and predicted as pathogenic (p.R57W, p.G68S, and p.R265C). Given that rare mutations with high penetrance contribute to schizophrenia, missense mutations of GNB1L might increase the risk of schizophrenia in some patients.

Multiple Rare Risk Coding Variants in Postsynaptic Density-Related Genes Associated With Schizophrenia Susceptibility.

OBJECTIVE: Schizophrenia is a chronic debilitating neurobiological disorder of aberrant synaptic connectivity and synaptogenesis. Postsynaptic density (PSD)-related proteins in N-methyl-D-aspartate receptor-postsynaptic signaling complexes are crucial to regulating the synaptic transmission and functions of various synaptic receptors. This study examined the role of PSD-related genes in susceptibility to schizophrenia. METHODS: We resequenced 18 genes encoding the disks large-associated protein (DLGAP), HOMER, neuroligin (NLGN), neurexin, and SH3 and multiple ankyrin repeat domains (SHANK) protein families in 98 schizophrenic patients with family psychiatric history using semiconductor sequencing. We analyzed the protein function of the identified rare schizophrenia-associated mutants via immunoblotting and immunocytochemistry. RESULTS: We identified 50 missense heterozygous mutations in 98 schizophrenic patients with family psychiatric history, and in silico analysis revealed some as damaging or pathological to the protein function. Ten missense mutations were absent from the dbSNP database, the gnomAD (non-neuro) dataset, and 1,517 healthy controls from Taiwan BioBank. Immunoblotting revealed eight missense mutants with altered protein expressions in cultured cells compared with the wild type. CONCLUSION: Our findings suggest that PSD-related genes, especially the NLGN, SHANK, and DLGAP families, harbor rare functional mutations that might alter protein expression in some patients with schizophrenia, supporting contributing rare coding variants into the genetic architecture of schizophrenia.

Genetic analysis of the NR2E1 gene as a candidate gene of schizophrenia.

NR2E1 is implicated in the regulation of neurogenesis and considered as a candidate gene for schizophrenia. We resequenced all the exons of NR2E1 in 547 patients with schizophrenia and 567 controls from Taiwan. We identified five common SNPs with no association with patients with schizophrenia. Further haplotype-based association analysis showed that two haplotypes within NR2E1 were correlated with the schizophrenia risk. Four rare mutations located at untranslated regions were identified in patients with schizophrenia but not in our control sample. The present study suggests that NR2E1 is likely to play a significant role in conferring susceptibility to schizophrenia.

Differential Expression of Multiple Disease-Related Protein Groups Induced by Valproic Acid in Human SH-SY5Y Neuroblastoma Cells.

Valproic acid (VPA) is a multifunctional medication used for the treatment of epilepsy, mania associated with bipolar disorder, and migraine. The pharmacological effects of VPA involve a variety of neurotransmitter and cell signaling systems, but the molecular mechanisms underlying its clinical efficacy is to date largely unknown. In this study, we used the isobaric tags for relative and absolute quantitation shotgun proteomic analysis to screen differentially expressed proteins in VPA-treated SH-SY5Y cells. We identified changes in the expression levels of multiple proteins involved in Alzheimer's disease, Parkinson's disease, chromatin remodeling, controlling gene expression via the vitamin D receptor, ribosome biogenesis, ubiquitin-mediated proteolysis, and the mitochondrial oxidative phosphorylation and electron transport chain. Our data indicate that VPA may modulate the differential expression of proteins involved in mitochondrial function and vitamin D receptor-mediated chromatin transcriptional regulation and proteins implicated in the pathogenesis of neurodegenerative diseases.

Functional analyses and effect of DNA methylation on the EGR1 gene in patients with schizophrenia.

EGR1, involved in the regulation of synaptic plasticity, learning, and memory, is considered a candidate gene for schizophrenia. We resequenced the exonic regions of EGR1 in 516 patients with schizophrenia and conducted a reporter gene assay. We found two mutations including a rare mutation (c.-8C>T, rs561524195) and one common SNP (c.308-42C>T, rs11743810). The reporter gene assay showed c.-8C>T mutant did not affect promoter activity. Gene expression analyses showed that the average EGR1 mRNA and protein levels in lymphoblastoid cell lines of schizophrenia in male, but not female, were significantly higher than those in controls. We conducted in vitro DNA methylation reaction, luciferase activity assay, and pyrosequencing to assess DNA methylation of EGR1 expression underlying the pathophysiology of schizophrenia. DNA methylation of the EGR1 promoter region attenuated reporter activity, suggesting that DNA methylation regulates EGR1 expression. There were no statistically significant differences in DNA methylation levels of 17 CpG sites at the EGR1 promoter region between 64 patients with schizophrenia compared with 64 controls. These results suggest that the exonic mutations in EGR1 and DNA methylation regulating EGR1 expression might not be associated with schizophrenia. However, the gender-specific association of elevated EGR1 expression might be involved in the pathophysiology of schizophrenia.

Mutation analysis of the WNT7A gene in patients with schizophrenia.

Aberrant WNT signaling has been implicated in the pathophysiology of schizophrenia. WNT7A, a member of the WNT gene family, is considered a potential candidate of schizophrenia. All exons of WNT7A in 570 schizophrenic patients and 563 controls were sequenced, and protein functional analysis was conducted. Five common variants were identified, but none were noted to be associated with schizophrenia. Nevertheless, nine rare mutations, including one schizophrenia-specific missense mutation (c.305G > A), were discovered. However, immunoblot analysis findings revealed that the c.305G > A mutation did not affect protein expression. These results suggest that WNT7A is unlikely to be associated with susceptibility to schizophrenia.

DNA methylation analysis of the EGR3 gene in patients of schizophrenia.

DNA methylation has been implicated in the pathogenesis of schizophrenia. EGR3 is considered as a potential candidate gene for schizophrenia. We conducted in vitro DNA methylation reaction, Lucia luciferase activity assay, and pyrosequencing assay to assess the DNA methylation of the EGR3 expression underlying the pathophysiology of schizophrenia. We found that DNA methylation of the putative EGR3 regulatory regions attenuated Lucia luciferase activity. There was no difference in the DNA methylation pattern of EGR3 between in 50 schizophrenic patients and 47 controls. Our data suggest that DNA methylation regulated the expression of EGR3 might not be associated with schizophrenia.

Screening for Mutations in the TBX1 Gene on Chromosome 22q11.2 in Schizophrenia.

A higher-than-expected frequency of schizophrenia in patients with 22q11.2 deletion syndrome suggests that chromosome 22q11.2 harbors the responsive genes related to the pathophysiology of schizophrenia. The TBX1 gene, which maps to the region on chromosome 22q11.2, plays a vital role in neuronal functions. Haploinsufficiency of the TBX1 gene is associated with schizophrenia endophenotype. This study aimed to investigate whether the TBX1 gene is associated with schizophrenia. We searched for mutations in the TBX1 gene in 652 patients with schizophrenia and 567 control subjects using a re-sequencing method and conducted a reporter gene assay. We identified six SNPs and 25 rare mutations with no association with schizophrenia from Taiwan. Notably, we identified two rare schizophrenia-specific mutations (c.-123G>C and c.-11delC) located at 5' UTR of the TBX1 gene. The reporter gene assay showed that c.-123C significantly decreased promoter activity, while c.-11delC increased promoter activity compared with the wild-type. Our findings suggest that the TBX1 gene is unlikely a major susceptible gene for schizophrenia in an ethnic Chinese population for Taiwan, but a few rare mutations in the TBX1 gene may contribute to the pathogenesis of schizophrenia in some patients.

Rare mutations and hypermethylation of the ARC gene associated with schizophrenia.

Activity-regulated cytoskeleton-associated protein (ARC), which interacts with the N-methyl-d-aspartate receptor (NMDAR) complex, is a critical effector molecule downstream of multiple neuronal signaling pathways. Dysregulation of the ARC/NMDAR complex can disrupt learning, memory, and normal brain functions. This study examined the role of ARC in susceptibility to schizophrenia. We used a resequencing strategy to identify the variants of ARC in 1078 subjects, including patients with schizophrenia and normal controls. We identified 16 known SNPs and 27 rare mutations. SNP-based analysis showed no association of ARC with schizophrenia. In addition, the rare mutations did not increase the burden in patients compared with controls. However, one patient-specific allele in the putative ARC promoter region and seven patient-specific mutants in ARC exon regions significantly reduced the reporter gene activity compared with ARC wild-type. Methylation of a putative ARC promoter attenuated reporter activity in vitro, suggesting that ARC expression is regulated by DNA methylation. Pyrosequencing revealed eight hypermethylated CpG sites in the putative ARC promoter region in 64 schizophrenic patients compared with 63 controls. Taken together, our results suggest that both rare variants and epigenetic regulation of ARC contribute to the pathogenesis of schizophrenia in some patients.

Chronic methamphetamine treatment reduces the expression of synaptic plasticity genes and changes their DNA methylation status in the mouse brain.

Methamphetamine (METH) is a highly addictive psychostimulant that may cause long-lasting synaptic dysfunction and abnormal gene expression. We aimed to explore the differential expression of synaptic plasticity genes in chronic METH-treated mouse brain. We used the RT(2) Profiler PCR Array and the real-time quantitative PCR to characterize differentially expressed synaptic plasticity genes in the frontal cortex and the hippocampus of chronic METH-treated mice compared with normal saline-treated mice. We further used pyrosequencing to assess DNA methylation changes in the CpG region of the five immediate early genes (IEGs) in chronic METH-treated mouse brain. We detected six downregulated genes in the frontal cortex and the hippocampus of chronic METH-treated mice, including five IEGs (Arc, Egr2, Fos, Klf10, and Nr4a1) and one neuronal receptor gene (Grm1), compared with normal saline-treated group, but only four genes (Arc, Egr2, Fos, and Nr4a1) were confirmed to be different. Furthermore, we found several CpG sites of the Arc and the Fos that had significant changes in DNA methylation status in the frontal cortex of chronic METH-treated mice, while the klf10 and the Nr4a1 that had significant changes in the hippocampus. Our results show that chronic administration of METH may lead to significant downregulation of the IEGs expression in both the frontal cortex and the hippocampus, which may partly account for the molecular mechanism of the action of METH. Furthermore, the changes in DNA methylation status of the IEGs in the brain indicate that an epigenetic mechanism-dependent transcriptional regulation may contribute to METH addiction, which warrants additional study.

Resequencing of early growth response 2 (EGR2) gene revealed a recurrent patient-specific mutation in schizophrenia.

Abnormal myelination is considered as part of the pathophysiology of schizophrenia. We resequenced the genomic DNA of the EGR2, which has a specific function in the myelination of peripheral nervous system, in 543 schizophrenic patients and 554 non-psychotic controls. We identified six known SNPs, which were not associated with schizophrenia. Nevertheless, we discovered 24 rare mutations, some of them were patient-specific, including a recurrent mutation (p.P173_Y174insP), which might be associated with the pathogenesis of schizophrenia.

Role of the DLGAP2 gene encoding the SAP90/PSD-95-associated protein 2 in schizophrenia.

Aberrant synaptic dysfunction is implicated in the pathogenesis of schizophrenia. The DLGAP2 gene encoding the SAP90/PSD-95-associated protein 2 (SAPAP2) located at the post-synaptic density of neuronal cells is involved in the neuronal synaptic function. This study aimed to investigate whether the DLGAP2 gene is associated with schizophrenia. We resequenced the putative promoter region and all the exons of the DLGAP2 gene in 523 patients with schizophrenia and 596 non-psychotic controls from Taiwan and conducted a case-control association analysis. We identified 19 known SNPs in this sample. Association analysis of 9 SNPs with minor allele frequency greater than 5% showed no association with schizophrenia. However, we found a haplotype (CCACCAACT) significantly associated with schizophrenia (odds ratio:2.5, p<0.001). We also detected 16 missense mutations and 1 amino acid-insertion mutation in this sample. Bioinformatic analysis showed some of these mutations were damaging or pathological to the protein function, but we did not find increased burden of these mutations in the patient group. Notably, we identified 5 private rare variants in 5 unrelated patients, respectively, including c.-69+9C>T, c.-69+13C>T, c.-69+47C>T, c.-69+55C>T at intron 1 and c.-32A>G at untranslated exon 2 of the DLGAP2 gene. These rare variants were not detected in 559 control subjects. Further reporter gene assay of these rare variants except c.-69+13C>T showed significantly elevated promoter activity than the wild type, suggesting increased DLGAP2 gene expression may contribute to the pathogenesis of schizophrenia. Our results indicate that DLGAP2 is a susceptible gene of schizophrenia.

Effect of clustering on ellipsometric spectra of randomly distributed gold nanoparticles on a substrate.

We present a theoretical model for describing light scattering from randomly distributed Au nanoparticles on a substrate, including the clustering effect. By using the finite-element Green's function method and spherical harmonic basis functions, we are able to calculate the polarization-dependent reflectivity spectra of the system (modeled by randomly distributed nanoparticles coupled with clusters) efficiently and accurately. The calculated ellipsometric spectra of the system with clusters can adequately describe the experimental data for the whole frequency range. We find that the clustering effect leads to some prominent features in the low frequency range of the ellipsometric spectra, which are attributed to plasmonic resonances associated with the coupling of Au nanoparticles and clusters.

Exonic resequencing of the DLGAP3 gene as a candidate gene for schizophrenia.

We resequenced the exonic regions of the DLGAP3 gene, which encodes SAP90/PSD95-associated protein 3, in 215 schizophrenic patients and 215 non-psychotic controls. Seven known single-nucleotide polymorphisms (SNPs) were identified, but not associated with schizophrenia. Nevertheless, we identified several rare missense mutations and some of them might be associated with the pathogenesis of schizophrenia.

Genetic analysis of the DLGAP1 gene as a candidate gene for schizophrenia.

Schizophrenia is a severe chronic mental disorder with high genetic components in its etiology. Several studies indicated that synaptic dysfunction is involved in the pathophysiology of schizophrenia. Postsynaptic synapse-associated protein 90/postsynaptic density 95-associated proteins (SAPAPs) constitute a part of the N-methyl-d-aspartate receptor-associated postsynaptic density proteins, and are involved in synapse formation. We hypothesized that genetic variants of the SAPAPs might be associated with schizophrenia. Thus, we systemically sequenced all the exons of the discs, large (Drosophila) homolog-associated protein 1 (DLGAP1) gene that encodes SAPAP1 in a sample of 121 schizophrenic patients and 120 controls from Taiwan. We totally identified six genetic variants, including five known SNPs (rs145691437, rs3786431, rs201567254, rs3745051 and rs11662259) and one rare missense mutation (c.1922A>G) in this sample. SNP- and haplotype-based analyses showed no association of these SNPs with schizophrenia. The c.1922A>G mutation that changes the amino acid lysine to arginine at codon 641 was found in one out of 121 patients, but not in 275 control subjects, suggesting it might be a patient-specific mutation. Nevertheless, bioinformatic analysis showed this mutation does not affect the function of the DLGAP1 gene and appears to be a benign variant. Hence, its relationship with the pathogenesis remains to be investigated.